Variants of vascular endothelial cell growth factors having antagonistic properties, nucleic acids encoding the same and host cells comprising those nucleic acids

ABSTRACT

The present invention involves the preparation of vascular endothelial growth factor (VEGF) antagonist molecules comprising variant VEGF polypeptides which are capable of binding to and occupying cell surface VEGF receptors without inducing a VEGF response, thereby antagonizing the biological activity of the native VEGF protein. Specifically, the variant VEGF polypeptides of the present invention comprise modifications of at least one cysteine residue in the native VEGF sequence, thereby inhibiting the ability of the variant polypeptide to dimerize through the formation of disulfide bonds. The present invention is further directed to methods for preparing such variant VEGF antagonists and to methods, compositions and assays utilizing such variants for producing pharmaceutically active materials having therapeutic and pharmacologic properties that differ from the native VEGF protein.

This application is a continuation of application Ser. No. 10/793,094,filed Mar. 3, 2004, which is a divisional of application Ser. No.08/734,443, filed Oct. 17, 1996, now U.S. Pat. No. 6,750,044, whichapplications are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention is directed to particular variants of vascularendothelial cell growth factor (hereinafter sometimes referred to asVEGF) which bind to and occupy cell surface VEGF receptors withoutinducing a VEGF response, thereby antagonizing the biological activityof the native VEGF protein. The present invention is further directed tomethods for preparing such variant VEGF antagonists and to methods,compositions and assays utilizing such variants for producingpharmaceutically active materials having therapeutic and pharmacologicproperties that differ from the native VEGF protein.

BACKGROUND OF THE INVENTION

The two major cellular components of the mammalian vascular system arethe endothelial and smooth muscle cells. Endothelial cells form thelining of the inner surface of all blood vessels in the mammal andconstitute a non-thrombogenic interface between blood and tissue.Therefore, the proliferation of endothelial cells is an importantcomponent for the development of new capillaries and blood vesselswhich, in turn, is a necessary process for the growth and/orregeneration of mammalian tissues.

One protein that has been shown to play an extremely important role inpromoting endothelial cell proliferation and angiogenesis is vascularendothelial cell growth factor (VEGF). VEGF is a heparin-bindingendothelial cell-specific growth factor which was originally identifiedand purified from media conditioned by bovine pituitary follicular orfolliculostellate (FS) cells. Ferrara and Henzel, Biochem. Biophys. Res.Comm. 161:851-858 (1989). Naturally-occurring VEGF is a dimeric proteinhaving an apparent molecular mass of about 46 kDa with each subunithaving an apparent molecular mass of about 23 kDa. Normal dimerizationbetween individual native VEGF monomers occurs through the formation ofdisulfide bonds between the cysteine residues located at amino acidposition 51 of one monomeric unit bonding to the cysteine residue atamino acid position 60 of another monomeric unit and vice versa. HumanVEGF is expressed in a variety of tissues as multiple homodimeric forms(121, 165, 189 and 206 amino acids per monomer), wherein each formarises as a result of alternative splicing of a single RNA transcript.For example, VEGF₁₂₁ is a soluble mitogen that does not bind heparinwhereas the longer forms of VEGF bind heparin with progressively higheraffinity.

Biochemical analyses have shown that the native VEGF dimer exhibits astrong mitogenic specificity for vascular endothelial cells. Forexample, media conditioned by cells transfected by human VEGF cDNApromoted the proliferation of capillary endothelial cells, whereasmedium conditioned by control cells did not. Leung et al., Science246:1306 (1989). Thus, the native VEGF dimer is known to promotevascular endothelial cell proliferation and angiogenesis, a processwhich involves the formation of new blood vessels from preexistingendothelium. As such, the native VEGF may be useful for the therapeutictreatment of numerous conditions in which a growth-promoting activity onthe vascular endothelial cells is important, for example, in ulcers,vascular injuries and myocardial infarction.

The endothelial cell proliferative activity of the VEGF dimer is knownto be mediated by two high affinity tyrosine kinase receptors, fit-1(FMS-like tyrosine kinase) and KDR (kinase domain region), which existonly on the surface of vascular endothelial cells. DeVries, et al.,Science 225:989-991 (1992) and Terman, et al., Oncogene 6:1677-1683(1991). As cells become depleted in oxygen, because of trauma and thelike, VEGF production increases in such cells, wherein the generatedVEGF protein subsequently binds to its respective cell surface receptorsin order to signal ultimate biological effect. The signal then increasesvascular permeability and the cells divide and expand to form newvascular pathways. Thus, native VEGF functions to induce vascularproliferation through the binding to endothelial cell-specificreceptors.

While VEGF-induced vascular endothelial cell proliferation is desirableunder certain circumstances, vascular endothelial cell proliferation andangiogenesis are also important components of a variety of diseases anddisorders. Such diseases and disorders include tumor growth andmetastasis, rheumatoid arthritis, psoriasis, atherosclerosis, diabeticretinopathy, retrolental fibroplasia, neovascular glaucoma, age-relatedmacular degeneration, hemangiomas, immune rejection of transplantedcorneal tissue and other tissues, and chronic inflammation. Obviously,in individuals suffering from any of these disorders, one would want tohave a means for inhibiting, or at least substantially reducing, theendothelial cell proliferating activity of the native VEGF dimericprotein.

Having an available means for inhibiting native VEGF activity isimportant for a number of reasons. For example, in the specific case oftumor cell growth, angiogenesis appears to be crucial for the transitionfrom hyperplasia to neoplasia and for providing nourishment to thegrowing solid tumor. Folkman, et al., Nature 339:58 (1989). Angiogenesisalso allows tumors to be in contact with the vascular bed of the host,which may provide a route for metastasis of tumor cells. Evidence forthe role of angiogenesis in tumor metastasis is provided, for example,by studies-showing a correlation between the number and density ofmicrovessels in histologic sections of invasive human breast carcinomaand actual presence of distant metastasis. Weidner et al., New Engl. J.Med. 324:1 (1991). Thus, one possible mechanism for the effectivetreatment of neoplastic tumors is to inhibit or substantially reduce theendothelial cell proliferative and angiogenic activity of the nativedimeric VEGF protein.

Therefore, in view of the role that VEGF-induced vascular endothelialcell growth and angiogenesis play in many diseases and disorders, it isdesirable to have a means for reducing or substantially inhibiting oneor more of the biological effects of the native VEGF protein, forexample, the mitogenic or angiogenic effect thereof. Thus, the presentinvention is predicated upon research intended to identify novel VEGFvariant polypeptides which are capable of inhibiting one or more of thebiological activities of native VEGF. Specifically, the presentinvention is predicated upon the identification of VEGF variants whichare capable of binding to and occupying cell-surface VEGF receptorswithout inducing a typical VEGF response, thereby effectively reducingor substantially inhibiting the effects of native VEGF. It waspostulated that if one could prepare such VEGF variants, one could usesuch variants in instances of tumor treatment in order to starve thetumors for intended regression.

It was a further object of this research to produce VEGF variants whichlose the ability to properly dimerize through the formation of covalentcysteine-cysteine disulfide bonds. Such variants include variant VEGFmonomers which lack the ability to dimerize through the formation ofcysteine-cysteine disulfide bonds and variant VEGF monomers which maydimerize through the formation of at least one cysteine-cysteinedisulfide bond, however, wherein at least one disulfide bond differsfrom that existing in the native VEGF dimer. Such variants possess theability to bind to and occupy cell surface VEGF receptors withoutinducing a VEGF response, thereby competing with native VEGF for bindingto the receptors and antagonistically inhibiting the biological activityof the native VEGF dimer.

As further objects, the VEGF variants of the present invention can beemployed in assays systems to discover small molecule agonists andantagonists for intended therapeutic use.

The results of the above described research is the subject of thepresent invention. We herein demonstrate that mutation or modificationof the cysteine residues at amino acid positions 51 and/or 60 of thenative VEGF amino acid sequence functions to produce VEGF variants whichlose the ability to properly dimerize. Specifically, substitution ofcysteine at positions 51 and/or 60 with another amino acid ormodification of the cysteine at that site prevents the ability of thatamino acid to participate in the formation of a disulfide bond. Thesevariants, however, retain the ability to bind to and occupy cell surfaceVEGF receptors without inducing a VEGF response, thereby effectivelyinhibiting the biological activity of the native VEGF dimer.

SUMMARY OF THE INVENTION

The present invention provides variants of the native VEGF protein whichare capable of binding to a VEGF receptor on the surface of vascularendothelial cells, thereby occupying those binding sites and inhibitingthe mitogenic, angiogenic or other biological activities of the nativeVEGF protein. The novel antagonist molecules of the present invention,therefore, are useful for the treatment of diseases or disorderscharacterized by undesirable excessive vascularization, including by wayof example, tumors, and especially solid malignant tumors, rheumatoidarthritis, psoriasis, atherosclerosis, diabetic and other retinopathies,retrolental fibroplasia, age-related macular degeneration, neovascularglaucoma, hemangiomas, thyroid hyperplasias (including Grave's disease),corneal and other tissue transplantation, and chronic inflammation. Theantagonists of the present invention are also useful for the treatmentof diseases or disorders characterized by undesirable vascularpermeability, such as edema associated with brain tumors, ascitesassociated with malignancies, Meigs' syndrome, lung inflammation,nephrotic syndrome, pericardial effusion (such as that associated withpericarditis) and pleural effusion.

In a preferred embodiment, the variant VEGF polypeptides of theantagonist molecules of the present invention comprise amino acidmodifications of at least one cysteine residue present in the nativeVEGF amino acid sequence wherein modification of that cysteineresidue(s) results in the polypeptide being incapable of properlydimerizing with another VEGF polypeptide.

In a particularly preferred embodiment, the cysteine residues of thenative VEGF amino acid sequence that are modified are at amino acidpositions 51 and/or 60 of the native VEGF amino acid sequence.

The novel VEGF variant polypeptides of the present invention may berecombinantly generated by creating at least one amino acid mutation ata cysteine residue in the native VEGF amino acid sequence such that thevariant is incapable of properly dimerizing. Typical mutations include,for example, substitutions, insertions and/or deletions. The cysteineresidue(s) of interest may also be chemically modified so as to beincapable of participating in a disulfide bond.

In other embodiments, the present invention is directed to isolatednucleic acid sequences encoding the novel VEGF antagonist molecules ofthe present invention and replicable expression vectors comprising thosenucleic acid sequences.

In still other embodiments, the present invention is directed to hostcells which are transfected with the replicable expression vectors ofthe present invention and are capable of expressing those vectors.

In yet another embodiment, the present invention is directed to acomposition for treating indications wherein anti-angiogenesis isdesired, such as in arresting tumor growth, comprising a therapeuticallyeffective amount of the antagonist molecule of the present inventioncompounded with a pharmaceutically acceptable carrier. Anotherembodiment of the present invention is directed to a method of treatingcomprising administering a therapeutically effective amount of the abovedescribed composition.

Expanding on the basic premise hereof of the discovery and mutagenesisof the native VEGF polypeptide to produce variant VEGF polypeptides, thepresent invention is directed to all associated embodiments derivingtherefrom, including recombinant DNA materials and processes forpreparing such variants, materials and information for compounding suchvariants into pharmaceutically finished form and assays using suchvariants to screen for candidates that have agonistic or antagonisticproperties with respect to the native VEGF polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict both the amino acid and DNA sequence for a nativeVEGF protein having 165 amino acids. Predicted amino acids of theprotein are shown below the DNA sequence and are numbered from the firstresidue of the N-terminus of the protein sequence. Negative amino acidnumbers refer to the presumed leader signal sequence or pre-protein,while positive numbers refer to the putative mature protein.

FIG. 2 is a schematic diagram showing the native VEGF dimer moleculehaving disulfide bonds between cysteine residues at amino acid positions51 and 60 and 60 and 51, respectively, of the monomeric units, variantpolypeptide C51D, wherein the cysteine residue at amino acid position 51has been substituted by an aspartic acid residue resulting in theformation of a staggered dimer, variant polypeptide C60D, wherein thecysteine residue at amino acid position 60 has been substituted by anaspartic acid residue resulting in the formation of a staggered dimerand variant polypeptide C51D, C60D, wherein the cysteine residues atboth amino acid positions 51 and 60 have been substituted by asparticacid residues, thereby preventing disulfide bond formation anddimerization.

FIG. 3 is a graph showing the binding profiles of native VEGF dimer(“•”), the staggered dimer formed from the C60D variant VEGF polypeptide(“□”), the staggered dimer formed from the C51D variant VEGF polypeptide(“∘”) and the monomeric VEGF variant polypeptide C51D, C60D (“Δ”) to theKDR receptor. Data is presented as the ratio of bound polypeptide tofree versus the picomolar (pM) concentration of unlabeled competitor.

FIG. 4 is a graph showing the binding profiles of native VEGF dimer(“•”) and the monomeric VEGF variant polypeptide C51D, C60D (“▴”) to theKDR receptor. Data is presented as the ratio of bound polypeptide tofree versus the nanomolar (nM) concentration of unlabeled VEGFcompetitor.

FIG. 5 is a graph showing the binding profiles of native VEGF dimer(“•”), the staggered dimer formed from the C60D variant VEGF polypeptide(“▪”), the staggered dimer formed from the C51D variant VEGF polypeptide(“∘”) and the monomeric VEGF variant polypeptide C51D, C60D (“▴”) to theFLT-1 receptor. Data is presented as the ratio of bound polypeptide tofree versus the nanomolar (nM) concentration of unlabeled VEGFcompetitor.

FIG. 6 is a graph showing the binding profiles of native VEGF dimer(“•”) and the monomeric VEGF variant polypeptide C51D, C60D (“▪”) to theFLT-1 receptor. Data is presented as the ratio of bound polypeptide tofree versus the nanomolar (nM) concentration of unlabeled VEGFcompetitor.

FIG. 7 is a graph demonstrating the ability of the native VEGF dimer(“•”) the staggered dimer formed from the C60D variant VEGF polypeptide(“∘”), the staggered dimer formed from the C51D variant VEGF polypeptide(“Δ”) and the monomeric VEGF variant polypeptide C51D, C60D (“□”) tostimulate mitogenesis in endothelial cells. Data is presented as thetotal number of endothelial cells versus the picomolar (pM)concentration of polypeptide employed.

FIG. 8 is a graph demonstrating the ability of the anti-VEGF monoclonalantibody A461 (“▪”) and the monomeric VEGF variant polypeptide C51D,C60D (“•”) to inhibit VEGF-induced growth of endothelial cells. Data ispresented as the total number of endothelial cells versus the ratio ofantibody or monomer inhibitor to VEGF employed.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “vascular endothelial cell growth factor,” or “VEGF,”refers to a native mammalian growth factor as defined in U.S. Pat. No.5,332,671, including the human amino acid sequence shown in FIG. 1 andnaturally occurring allelic and processed forms of such growth factors.VEGF proteins can exist in either monomeric or multimeric (for example,dimeric) form. “Proper dimerization” is the dimerization which normallyoccurs between native VEGF monomers.

The term “native” with regard to a VEGF protein refers to a naturallyoccurring VEGF protein of any human or non-human animal species, with orwithout the initiating methionine, whether purified from the nativesource, synthesized, produced by recombinant DNA technology or by anycombination of these and/or other methods. Native VEGF proteinsnaturally exist as dimeric molecules, wherein the monomeric unitsthereof are covalently connected through the formation ofcysteine-cysteine disulfide bonds. Native VEGF specifically includes thenative human VEGF protein having the amino acid sequence shown in FIG. 1and possesses the ability to induce the proliferation of vascularendothelial cells in vivo.

The term “variant” with respect to a VEGF protein refers to a VEGFprotein that possesses at least one amino acid mutation or modification(i.e., alteration) as compared to a native VEGF protein and which may ormay not lack one or more of the biological activities of a native VEGFprotein. Variant VEGF proteins generated by “amino acid modifications”can be produced, for example, by substituting, deleting, insertingand/or chemically modifying at least one amino acid in the native VEGFamino acid sequence. Methods for creating such VEGF variants aredescribed below.

The term “monomeric variant”, “monomeric antagonist” or grammaticalequivalents thereof refers to a variant VEGF protein having at least oneamino acid alteration as compared to a native VEGF monomer, wherein saidamino acid alteration acts to prevent dimer formation between themonomeric units. Thus, the “monomeric variants” or “monomericantagonists” of the present invention are those VEGF variants which areincapable of dimerizing through the formation of cysteine-cysteinedisulfide bonds. Monomeric variants of the native VEGF protein, however,will possess the ability to bind to and occupy cell-surface VEGFreceptors without inducing a mitogenic and/or angiogenic VEGF response,although the binding affinity of the monomeric variant at thosereceptors may differ from that of a native VEGF protein.

The term “staggered dimer”, “staggered antagonist” or grammaticalequivalents thereof refers to a variant VEGF protein having at least oneamino acid alteration as compared to a native VEGF protein and whichretains the ability to dimerize through the formation of at least onecysteine-cysteine disulfide bond, however, where at least one of thedisulfide bonds formed is different from that which exists in the nativeVEGF dimeric protein.

A “functional derivative” of a polypeptide is a compound having aqualitative biological activity, or lack thereof, in common with theanother polypeptide. Thus, for example, a functional derivative of aVEGF antagonist compound of the present invention is a compound that hasa qualitative biological activity in common with an original polypeptideantagonist, for example, as being capable of binding to cell surfaceVEGF receptors without inducing a VEGF response, thereby occupying thosereceptors and inhibiting native VEGF activity. “Functional derivatives”include, but are not limited to, amino acid sequence variants of thevariant VEGF proteins of the present invention, fragments ofpolypeptides from any animal species (including humans), derivatives ofhuman and non-human polypeptides and their fragments, and peptideanalogs of native polypeptides, provided that they have a biologicalactivity, or lack thereof, in common with a respective variant VEGFprotein. “Fragments” comprise regions within the sequence of a maturepolypeptide. The term “derivative” is used to define amino acid sequencevariants, and covalent modifications of a polypeptide.

“Identity” or “homology” with respect to a polypeptide and/or itsfunctional derivatives is defined herein as the percentage of amino acidresidues in the candidate sequence that are identical with the residuesof a corresponding polypeptide, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology,and not considering any conservative substitutions as part of thesequence identity. Neither N- or C-terminal extensions nor insertionsshall be construed as reducing identity or homology. Methods andcomputer programs for the alignment are well known in the art.

The term “biological activity” in the context of the definition offunctional derivatives is defined as the possession of at least onefunction qualitatively in common with another polypeptide. Thefunctional derivatives of the polypeptide antagonists of the presentinvention are unified by their qualitative ability to bind to a VEGFreceptor without inducing a VEGF response, thereby preventing nativeVEGF from binding at that site and, in turn, inhibiting the biologicalactivity of the native VEGF protein.

The term “antagonist” is used to refer to a molecule inhibiting abiological activity of a native VEGF protein. Preferably, the VEGFantagonist compounds herein inhibit the ability of VEGF to inducevascular endothelial cell proliferation. Preferred antagonistsessentially completely inhibit vascular endothelial cell proliferation.

Ordinarily, the terms “amino acid” and “amino acids” refer to allnaturally occurring L-α-amino acids. In some embodiments, however,either D-amino acids or non-natural substituted amino acids may bepresent in the polypeptides or peptides of the present invention inorder to facilitate conformational restriction. For example, in order tofacilitate disulfide bond formation and stability, a D-amino acidcysteine may be provided at one or both termini of a peptide functionalderivative or peptide antagonist of the native VEGF protein. The aminoacids are identified by either the single-letter or three-letterdesignations: Asp D Aspartic acid Thr T Threonine Ser S Serine Glu EGlutamic acid Pro P Praline Gly G Glycine Ala A Alanine Cys C CysteineVal V Valine Met M Methionine IIe I Isoleucine Leu L Leucine Tyr YTyrosine Phe F Phenylalanine His H Histidine Lys K Lysine Arg R ArginineTrp W Tryptophan Gln Q Glutamine Asn N asparagineThese amino acids may be classified according to the chemicalcomposition and properties of their side chains. They are broadlyclassified into two groups, charged and uncharged. Each of these groupsis divided into subgroups to classify the amino acids more accurately:I. Charged Amino Acids

-   -   Acidic Residues: aspartic acid, glutamic acid    -   Basic Residues: lysine, arginine, histidine        II. Uncharged Amino Acids    -   Hydrophilic Residues: serine, threonine, asparagine, glutamine    -   Aliphatic Residues: glycine, alanine, valine, leucine,        isoleucine    -   Non-polar Residues: cysteine, methionine, proline    -   Aromatic Residues: phenylalanine, tyrosine, tryptophan

The term “amino acid sequence variant” or “amino acid alteration” refersto molecules having at least one differences in their amino acidsequence as compared to another amino acid sequence, usually the nativeamino acid sequence.

“Substitutional” variants are those that have at least one amino acidresidue in a corresponding sequence removed and a different amino acidinserted in its place at the same position. The substitutions may besingle, where only one amino acid in the molecule has been substituted,or they may be multiple, where two or more amino acids have beensubstituted in the same molecule.

“Insertional” variants are those with one or more amino acids insertedimmediately adjacent to an amino acid at a particular position in acorresponding sequence. Immediately adjacent to an amino acid meansconnected to either the α-carboxy or α-amino functional group of theamino acid.

“Deletional” variants are those with one or more amino acids in acorresponding amino acid sequence removed. Ordinarily, deletionalvariants will have one or two amino acids deleted in a particular regionof the molecule.

The term “isolated” means that a nucleic acid or polypeptide isidentified and separated from contaminant nucleic acids or polypeptidespresent in the animal or human source of the nucleic acid orpolypeptide.

Hybridization is preferably performed under “stringent conditions” whichmeans (1) employing low ionic strength and high temperature for washing,for example, 0.015 sodium chloride/0.0015 M sodium citrate/0.1% sodiumdodecyl sulfate at 50° C., or (2) employing during hybridization adenaturing agent, such as formamide, for example, 50% (vol/vol)formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 nM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C. Another example is useof 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6/8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 4° C. in 0.2×SSC and 0.1% SDS.Yet another example is hybridization using a buffer of 10% dextransulfate, 2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C., followed by a high-stringency wash consisting of 0.1×SSC containingEDTA at 55° C.

“Transfection” refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed.

Numerous methods of transfection are known to the ordinarily skilledartisan, for example, CaPO₄ and electroporation. Successful transfectionis generally recognized when any indication of the operation of thisvector occurs within the host cell.

“Transformation” means introducing DNA into an organism so that the DNAis replicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described by Cohen, S, N. Proc. Natl.Acad. Sci. (USA), 69, 2110 (1972) and Mandel et al. J. Mol. Biol. 53,154 (1970), is generally used for prokaryotes or other cells thatcontain substantial cell-wall barriers. For mammalian cells without suchcell walls, the calcium phosphate precipitation method of Graham, F. andvan der Eb, A., Virology, 52, 456-457 (1978) is preferred. Generalaspects of mammalian cell host system transformations have beendescribed by Axel in U.S. Pat. No. 4,399,216 issued Aug. 16, 1983.Transformations into yeast are typically carried out according to themethod of Van Solingen, P., et al. J. Bact., 130, 946 (1977) and Hsiao,C. L., et al. Proc. Natl. Acad. Sci. (USA) 76, 3829 (1979). However,other methods for introducing DNA into cells such as by nuclearinjection or by protoplast fusion may also be used.

“Site-directed mutagenesis” is a technique standard in the art, and isconducted using a synthetic oligonucleotide primer complementary to asingle-stranded phage DNA to be mutagenized except for limitedmismatching, representing the desired mutation. Briefly, the syntheticoligonucleotide is used as a primer to direct synthesis of a strandcomplementary to the single-stranded phage DNA, and the resultingdouble-stranded DNA is transformed into a phage-supporting hostbacterium. Cultures of the transformed bacteria are plated in top agar,permitting plaque formation from single cells that harbor the phage.Theoretically, 50% of the new plaques will contain the phage having, asa single strand, the mutated form; 50% will have the original sequence.Plaques of interest are selected by hybridizing with kinased syntheticprimer at a temperature that permits hybridization of an exact match,but at which the mismatches with the original strand are sufficient toprevent hybridization. Plaques that hybridize with the probe are thenselected, sequenced and cultured, and the DNA is recovered.

“Operably linked” refers to juxtaposition such that the normal functionof the components can be performed. Thus, a coding sequence “operablylinked” to control sequences refers to a configuration wherein thecoding sequence can be expressed under the control of these sequencesand wherein the DNA sequences being linked are contiguous and, in thecase of a secretory leader, contiguous and in reading phase. Forexample, DNA for a presequence or secretory leader is operably linked toDNA for a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation.Linking is accomplished by ligation at convenient restriction sites. Ifsuch sites do not exist, then synthetic oligonucleotide adaptors orlinkers are used in accord with conventional practice.

“Control sequences” refers to DNA sequences necessary for the expressionof an operably linked coding sequence in a particular host organism. Thecontrol sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, a ribosome bindingsite, and possibly, other as yet poorly understood sequences. Eukaryoticcells are known to utilize promoters, polyadenylation signals, andenhancers.

“Expression system” refers to DNA sequences containing a desired codingsequence and control sequences in operable linkage, so that hoststransformed with these sequences are capable of producing the encodedproteins. To effect transformation, the expression system may beincluded on a vector; however, the relevant DNA may then also beintegrated into the host chromosome.

As used herein, “cell,” “cell line,” and “cell culture” are usedinterchangeably and all such designations include progeny. Thus,“transformants” or “transformed cells” includes the primary subject celland cultures derived therefrom without regard for the number oftransfers. It is also understood that all progeny may not be preciselyidentical in DNA content, due to deliberate or inadvertent mutations.Mutant progeny that have the same functionality as screened for in theoriginally transformed cell are included. Where distinct designationsare intended, it will be clear from the context.

“Plasmids” are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein arecommercially available, are publicly available on an unrestricted basis,or can be constructed from such available plasmids in accord withpublished procedures. In addition, other equivalent plasmids are knownin the art and will be apparent to the ordinary artisan.

Digestion” of DNA refers to catalytic cleavage of the DNA with an enzymethat acts only at certain locations in the DNA. Such enzymes are calledrestriction enzymes, and the sites for which each is specific is calleda restriction site. The various restriction enzymes used herein arecommercially available and their reaction conditions, cofactors, andother requirements as established by the enzyme suppliers are used.Restriction enzymes commonly are designated by abbreviations composed ofa capital letter followed by other letters representing themicroorganism from which each restriction enzyme originally was obtainedand then a number designating the particular enzyme. In general, about 1mg of plasmid or DNA fragment is used with about 1-2 units of enzyme inabout 20 μl of buffer solution. Appropriate buffers and substrateamounts for particular restriction enzymes are specified by themanufacturer. Incubation of about 1 hour at 37° C. is ordinarily used,but may vary in accordance with the supplier's instructions. Afterincubation, protein is removed by extraction with phenol and chloroform,and the digested nucleic acid is recovered from the aqueous fraction byprecipitation with ethanol. Digestion with a restriction enzymeinfrequently is followed with bacterial alkaline phosphatase hydrolysisof the terminal 5′ phosphates to prevent the two restriction cleavedends of a DNA fragment from “circularizing” or forming a closed loopthat would impede insertion of another DNA fragment at the restrictionsite. Unless otherwise stated, digestion of plasmids is not followed by5′ terminal dephosphorylation. Procedures and reagents fordephosphorylation are conventional (T. Maniatis et al. 1982, MolecularCloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory,1982) pp. 133-134).

“Recovery” or “isolation” of a given fragment of DNA from a restrictiondigest means separation of the digest on polyacrylamide or agarose gelby electrophoresis, identification of the fragment of interest bycomparison of its mobility versus that of marker DNA fragments of knownmolecular weight, removal of the gel section containing the desiredfragment, and separation of the gel from DNA. This procedure is knowngenerally. For example, see R. Lawn et al., Nucleic Acids Res. 9,6103-6114 (1981), and D. Goeddel et al., Nucleic Acids Res. 8, 4057(1980).

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (T. Maniatis et al. 1982,supra, p. 146). Unless otherwise provided, ligation may be accomplishedusing known buffers and conditions with 10 units of T4 DNA ligase(“ligase”) per 0.5 mg of approximately equimolar amounts of the DNAfragments to be ligated.

“Preparation” of DNA from transformants means isolating plasmid DNA frommicrobial culture. Unless otherwise provided, the alkaline/SDS method ofManiatis et al. 1982, supra, p. 90, may be used.

“Oligonucleotides” are short-length, single- or double-strandedpolydeoxynucleotides that are chemically synthesized by known methods(such as phosphotriester, phosphite, or phosphoramidite chemistry, usingsolid phase techniques such as described in EP Pat. Pub. No. 266,032published May 4, 1988, or via deoxynucleoside H-phosphonateintermediates as described by Froehler et al., Nucl. Acids Res. 14,5399-5407 [1986]). They are then purified on polyacrylamide gels.

The abbreviation “KDR” refers to the kinase domain region of the VEGFmolecule, whether a native VEGF molecule or a variant thereof. It isthis region which is known to bind to the kinase domain region receptor.

The abbreviation “FLT-1” refers to the FMS-like tyrosine kinase bindingdomain which is known to bind to the corresponding flt-1 receptor. Thesereceptors exist on the surfaces of endothelial cells.

B. General Methodology

1. Glycosylation

The VEGF variants of the present invention may contain at least oneamino acid sequence that has the potential to be glycosylated through anN-linkage and that is not normally glycosylated in the native VEGFmolecule.

Introduction of an N-linked glycosylation site in the variant requires atripeptidyl sequence of the formula: asparagine-X-serine orasparagine-X-threonine, wherein asparagine is the acceptor and X is anyof the twenty genetically encoded amino acids except proline, whichprevents glycosylation. See D. K. Struck and W. J. Lennarz, in TheBiochemistry of Glycoproteins and Proteoglycans, ed. W. J. Lennarz,Plenum Press, 1980, p. 35; R. D. Marshall, Biochem. Soc. Symp., 40, 17(1974), and Winzler, R. J., in Hormonal Proteins and Peptides (ed. Li,C. I.) p. 1-15 (Academic Press, New York, 1973). The amino acid sequencevariant herein is modified by substituting for the amino acid(s) at theappropriate site(s) the appropriate amino acids to effect glycosylation.

If O-linked glycosylation is to be employed, O-glycosidic linkage occursin animal cells between N-acetylgalactosamine, galactose, or xylose andone of several hydroxyamino acids, most commonly serine or threonine,but also in some cases a 5-hydroxyproline or 5-hydroxylysine residueplaced in the appropriate region of the molecule.

Glycosylation patterns for proteins produced by mammals are described indetail in The Plasma Proteins: Structure, Function and Genetic Control,F. W. Putnam, ed., 2nd edition, volume 4 (Academic Press, New York,1984), p. 271-315, the entire disclosure of which is incorporated hereinby reference. In this chapter, asparagine-linked oligosaccharides arediscussed, including their subdivision into at least three groupsreferred to as complex, high mannose, and hybrid structures, as well asO-glucosidically linked oligosaccharides.

Chemical and/or enzymatic coupling of glycosides to proteins can beaccomplished using a variety of activated groups, for example, asdescribed by Aplin and Wriston in CRC Crit. Rev. Biochem., pp. 259-306(1981), the disclosure of which is incorporated herein by reference. Theadvantages of the chemical coupling techniques are that they arerelatively simple and do not need the complicated enzymatic machineryrequired for natural o- and N-linked glycosylation. Depending on thecoupling mode used, the sugar(s) may be attached to (a) arginine orhistidine, (b) free carboxyl groups such as those of glutamic acid oraspartic acid, (c) free sulfhydryl groups such as those of cysteine, (d)free hydroxyl groups such as those of serine, threonine, orhydroxyproline, (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan, or (f) the amide group of glutamine. Thesemethods are described more fully in PCT WO 87/05330 published Sep. 11,1987, the disclosure of which is incorporated herein by reference.

Glycosylation patterns for proteins produced by yeast are described indetail by Tanner and Lehle, Biochim. Biophys. Acta, 906(1), 81-99 (1987)and by Kukuruzinska et al., Annu. Rev. Biochem., 56, 915-944 (1987), thedisclosures of which are incorporated herein by reference.

2. Amino Acid Sequence Variants

a. Additional Mutations

For purposes of shorthand designation of the VEGF variants describedherein, it is noted that numbers refer to the amino acidresidue/position along the amino acid sequences of the putative matureVEGF protein shown in FIGS. 1A and 1B.

The present invention is directed to variants of VEGF where suchvariants have modifications in the amino acid sequence that affect theability of the VEGF monomeric units to properly dimerize. These variantshave the ability to bind to and occupy cell-surface VEGF receptorswithout substantially activating vascular endothelial proliferation andangiogenesis, thereby inhibiting the biological activity of native VEGF.Specifically, amino acid modifications can be made at amino acidpositions 51 and/or 60, each of which affect the ability of the variantVEGF monomers to properly dimerize. Moreover, additional variants basedupon these original variants can be made by means generally known wellin the art and without departing from the spirit of the presentinvention.

With regard to the VEGF variants of the present invention, for example,covalent modifications can be made to various of the amino acidresidues.

b. DNA Mutations

Amino acid sequence variants of VEGF and variants thereof can also beprepared by mutations in the DNA. Such variants include, for example,deletions from, or insertions or substitutions of, residues within theamino acid sequence shown in FIG. 1. Any combination of deletion,insertion, and substitution may also be made to arrive at the finalconstruct, provided that the final construct possesses the desiredactivity. Obviously, the mutations that will be made in the DNA encodingthe variant must not place the sequence out of reading frame andpreferably will not create complementary regions that could producesecondary mRNA structure (see EP 75,444A).

At the genetic level, these variants ordinarily are prepared bysite-directed mutagenesis of nucleotides in the DNA encoding the VEGF,thereby producing DNA encoding the variant, and thereafter expressingthe DNA in recombinant cell culture.

While the site for introducing an amino acid sequence variation ispredetermined, the mutation per se need not be predetermined. Forexample, to optimize the performance of a mutation at a given site,random mutagenesis may be conducted at the target codon or region andthe expressed VEGF variants screened for the optimal combination ofdesired activity. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence are well known, forexample, site-specific mutagenesis.

Preparation of VEGF variants in accordance herewith is preferablyachieved by site-specific mutagenesis of DNA that encodes an earlierprepared variant or a nonvariant version of the protein. Site-specificmutagenesis allows the production of VEGF variants through the use ofspecific oligonucleotide sequences that encode the DNA sequence of thedesired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 20 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered. In general, thetechnique of site-specific mutagenesis is well known in the art, asexemplified by publications such as Adelman et al., DNA 2, 183 (1983),the disclosure of which is incorporated herein by reference.

As will be appreciated, the site-specific mutagenesis techniquetypically employs a phage vector that exists in both a single-strandedand double-stranded form. Typical vectors useful in site-directedmutagenesis include vectors such as the M13 phage, for example, asdisclosed by Messing et al., Third Cleveland Symposium on Macromoleculesand Recombinant DNA, Editor A. Walton, Elsevier, Amsterdam (1981), thedisclosure of which is incorporated herein by reference. These phage arereadily commercially available and their use is generally well known tothose skilled in the art. Alternatively, plasmid vectors that contain asingle-stranded phage origin of replication (Veira et al., Meth.Enzymol., 153, 3 [1987]) may be employed to obtain single-stranded DNA.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector that includeswithin its sequence a DNA sequence that encodes the relevant protein. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically, for example, by the method of Crea et al.,Proc. Natl. Acad. Sci. (USA), 75, 5765 (1978). This primer is thenannealed with the single-stranded protein-sequence-containing vector,and subjected to DNA-polymerizing enzymes such as E. coli polymerase IKlenow fragment, to complete the synthesis of the mutation-bearingstrand. Thus, a heteroduplex is formed wherein one strand encodes theoriginal non-mutated sequence and the second strand bears the desiredmutation. This heteroduplex vector is then used to transform appropriatecells such as JM101 cells and clones are selected that includerecombinant vectors bearing the mutated sequence arrangement.

After such a clone is selected, the mutated protein region may beremoved and placed in an appropriate vector for protein production,generally an expression vector of the type that may be employed fortransformation of an appropriate host.

c. Types of Mutations

Amino acid sequence deletions generally range from about 1 to 30residues, more preferably 1 to 10 residues, and typically arecontiguous.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions of from one residue to polypeptides of essentially unrestrictedlength, as well as intrasequence insertions of single or multiple aminoacid residues. Intrasequence insertions (i.e., insertions within themature VEGF sequence) may range generally from about 1 to 10 residues,more preferably 1 to 5. An example of a terminal insertion includes afusion of a signal sequence, whether heterologous or homologous to thehost cell, to the N-terminus of the variant VEGF molecule to facilitatethe secretion of variant VEGF from recombinant hosts.

The third group of variants are those in which at least one amino acidresidue in the VEGF molecule, and preferably only one, has been removedand a different residue inserted in its place. Such substitutionspreferably are made in accordance with the following Table 1 when it isdesired to modulate finely the characteristics of a VEGF molecule orvariant thereof. TABLE 1 Original Residue Exemplary Substitutions Ala(A) gly; ser Arg (R) lys Asn (N) gln; his Asp (D) glu Cys (C) ser Gln(Q) asn Glu (E) asp Gly (G) ala; pro His (H) asn; gln IIe (I) leu; valLeu (L) ile; val Lys (K) arg; gln; glu Met (M) leu; tyr; ile Phe (F)met; leu; tyr Ser (S) thr Thr (T) ser Trp (W) trp; phe Val (V) ile; leuSubstantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in Table1, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain. The substitutions thatin general are expected to produce the greatest changes in biologicalproperties will be those in which (a) glycine and/or proline issubstituted by another amino acid or is deleted or inserted; (b) ahydrophilic residue, e.g., seryl or threonyl, is substituted for (or by)a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, oralanyl; (c) a cysteine residue is substituted for (or by) any otherresidue; (d) a residue having an electropositive side chain, e.g.,lysyl, arginyl, or histidyl, is substituted for (or by) a residue havingan electronegative charge, e.g., glutamyl or aspartyl; (e) a residuehaving an electronegative side chain is substituted for (or by) aresidue having an electropositive charge; or (f) a residue having abulky side chain, e.g., phenylalanine, is substituted for (or by) onenot having such a side chain, e.g., glycine. Most deletions andinsertions, and substitutions in particular, are not expected to produceradical changes in the characteristics of the VEGF molecule or variantthereof. However, when it is difficult to predict the exact effect ofthe substitution, deletion, or insertion in advance of doing so, oneskilled in the art will appreciate that the effect will be evaluated byroutine screening assays. For example, a variant typically is made bysite-specific mutagenesis of the native VEGF-encoding nucleic acid,expression of the variant nucleic acid in recombinant cell culture, and,optionally, purification from the cell culture, for example, byimmunoaffinity adsorption on a rabbit polyclonal anti-VEGF column (toabsorb the variant by binding it to at least one remaining immuneepitope).

Since VEGF tends to aggregate into dimers, it is within the scope hereofto provide hetero- and homodimers, wherein one or both subunits arevariants. Where both subunits are variants, the changes in amino acidsequence can be the same or different for each subunit chain.Heterodimers are readily produced by cotransforming host cells with DNAencoding both subunits and, if necessary, purifying the desiredheterodimer, or by separately synthesizing the subunits, dissociatingthe subunits (e.g., by treatment with a chaotropic agent such as urea,guanidine hydrochloride, or the like), mixing the dissociated subunits,and then reassociating the subunits by dialyzing away the chaotropicagent.

Also included within the scope of mutants herein are so-calledglyco-scan mutants. This embodiment takes advantage of the knowledge ofso-called glycosylation sites which are identified by thesequence—NX(S/T) wherein N represents the amino acid asparagine, Xrepresents any amino acid except proline and probably glycine and thethird position can be occupied by either amino acid serine or threonine.Thus, where appropriate, such a glycosylation site can be introduced soas to produce a species containing glycosylation moieties at thatposition. Similarly, an existing glycosylation site can be removed bymutation so as to produce a species that is devoid of glycosylation atthat site. It will be understood, again, as with the other mutationscontemplated by the present invention, that they are introduced at aminoacid position(s) 51 and/or 60 of the native VEGF amino acid sequence inaccord with the basic premise of the present invention, and they can beintroduced at other locations outside of these amino acid positionswithin the overall molecule so long as the final product does not differin overall kind from the properties of the original VEGF variant.

The activity of the cell lysate or purified VEGF variant is thenscreened in a suitable screening assay for the desired characteristic.For example, binding to the cell-surface VEGF receptor can be routinelyassayed by employing well known VEGF binding assays such as thosedescribed in the Examples below. A change in the immunological characterof the VEGF molecule, such as affinity for a given antibody, is measuredby a competitive-type immunoassay. Changes in the enhancement orsuppression of vascular endothelium growth by the candidate variants aremeasured by the appropriate assay (see Examples below). Modifications ofsuch protein properties as redox or thermal stability, hydrophobicity,susceptibility to proteolytic degradation, or the tendency to aggregatewith carriers or into multimers are assayed by methods well known to theordinarily skilled artisan.

3. Recombinant Expression

The variant VEGF molecule desired may be prepared by any technique,including by recombinant methods. Likewise, an isolated DNA isunderstood herein to mean chemically synthesized DNA, cDNA, chromosomal,or extrachromosomal DNA with or without the 3′- and/or 5′-flankingregions. Preferably, the desired VEGF variant herein is made bysynthesis in recombinant cell culture.

For such synthesis, it is first necessary to secure nucleic acid thatencodes a VEGF molecule. DNA encoding a VEGF molecule may be obtainedfrom bovine pituitary follicular cells by (a) preparing a cDNA libraryfrom these cells, (b) conducting hybridization analysis with labeled DNAencoding the VEGF or fragments thereof (up to or more than 100 basepairs in length) to detect clones in the library containing homologoussequences, and (c) analyzing the clones by restriction enzyme analysisand nucleic acid sequencing to identify full-length clones. DNA encodinga VEGF molecule from a mammal other than bovine can also be obtained ina similar fashion by screening endothelial or leukemia cell libraries.DNA that is capable of hybridizing to a VEGF-encoding DNA under lowstringency conditions is useful for identifying DNA encoding VEGF. Bothhigh and low stringency conditions are defined further below. Iffull-length clones are not present in a cDNA library, then appropriatefragments may be recovered from the various clones using the nucleicacid sequence information disclosed herein for the first time andligated at restriction sites common to the clones to assemble afull-length clone encoding the VEGF molecule. Alternatively, genomiclibraries will provide the desired DNA.

Once this DNA has been identified and isolated from the library it isligated into a replicable vector for further cloning or for expression.

In one example of a recombinant expression system a VEGF-encoding geneis expressed in mammalian cells by transformation with an expressionvector comprising DNA encoding the VEGF. It is preferable to transformhost cells capable of accomplishing such processing so as to obtain theVEGF in the culture medium or periplasm of the host cell, i.e., obtain asecreted molecule.

a. Useful Host Cells and Vectors

The vectors and methods disclosed herein are suitable for use in hostcells over a wide range of prokaryotic and eukaryotic organisms.

In general, of course, prokaryotes are preferred for the initial cloningof DNA sequences and construction of the vectors useful in theinvention. For example, E. coli K12 strain MM 294 (ATCC No. 31,446) isparticularly useful. Other microbial strains that may be used include E.coli strains such as E. coli B and E. coli X1776 (ATCC No. 31,537).These examples are, of course, intended to be illustrative rather thanlimiting. Prokaryotes may also be used for expression. Theaforementioned strains, as well as E. coli strains W3110 (F-, lambda-,prototrophic, ATCC No. 27,325), K5772 (ATCC No. 53,635), and SR101,bacilli such as Bacillus subtilis, and other enterobacteriaceae such asSalmonella typhimurium or Serratia marcesans, and various pseudomonasspecies, may be used.

In general, plasmid vectors containing replicon and control sequencesthat are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences that are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies (see, e.g., Bolivar et al., Gene 2, 95 [1977]). pBR322 containsgenes for ampicillin and tetracycline resistance and thus provides easymeans for identifying transformed cells. The pBR322 plasmid, or othermicrobial plasmid or phage, must also contain, or be modified tocontain, promoters that can be used by the microbial organism forexpression of its own proteins.

Those promoters most commonly used in recombinant DNA constructioninclude the β-lactamase (penicillinase) and lactose promoter systems(Chang et al., Nature, 375, 615 [1978]; Itakura et al., Science, 198,1056 [1977]; Goeddel et al., Nature, 281, 544 [1979]) and a tryptophan(trp) promoter system (Goeddel et al., Nucleic Acids Res., 8, 4057[1980]; EPO Appl. Publ. No. 0036,776). While these are the most commonlyused, other microbial promoters have been discovered and utilized, anddetails concerning their nucleotide sequences have been published,enabling a skilled worker to ligate them functionally with plasmidvectors (see, e.g., Siebenlist et al., Cell, 20, 269 [1980]).

In addition to prokaryotes, eukaryotic microbes, such as yeast cultures,may also be used. Saccharomyces cerevisiae, or common baker's yeast, isthe most commonly used among eukaryotic microorganisms, although anumber of other strains are commonly available. For expression inSaccharomyces, the plasmid YRp7, for example (Stinchcomb et al., Nature282, 39 [1979]; Kingsman et al., Gene 7, 141 [1979]; Tschemper et al.,Gene 10, 157 [1980]), is commonly used. This plasmid already containsthe trp1 gene that provides a selection marker for a mutant strain ofyeast lacking the ability to grow in tryptophan, for example, ATCC No.44,076 or PEP4-1 (Jones, Genetics, 85, 12 [1977]). The presence of thetrp1 lesion as a characteristic of the yeast host cell genome thenprovides an effective environment for detecting transformation by growthin the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255, 2073[1980]) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7,149 [1968]; Holland et al., Biochemistry 17, 4900 [1978]), such asenolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also ligated into the expression vector 3′ of the sequencedesired to be expressed to provide polyadenylation of the mRNA andtermination. Other promoters, which have the additional advantage oftranscription controlled by growth conditions, are the promoter regionfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and theaforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Any plasmid vectorcontaining yeast-compatible promoter, origin of replication andtermination sequences is suitable.

In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) has become aroutine procedure in recent years [Tissue Culture, Academic Press, Kruseand Patterson, editors (1973)]. Examples of such useful host cell linesare VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, andW138, BHK, COS-7, 293, and MDCK cell lines. Expression vectors for suchcells ordinarily include (if necessary) an origin of replication, apromoter located in front of the gene to be expressed, along with anynecessary ribosome binding sites, RNA splice sites, polyadenylationsites, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expressionvectors are often provided by viral material. For example, commonly usedpromoters are derived from polyoma, Adenovirus2, and most frequentlySimian Virus 40 (SV40). The early and late promoters of SV40 virus areparticularly useful because both are obtained easily from the virus as afragment that also contains the SV40 viral origin of replication [Fierset al., Nature, 273, 113 (1978)]. Smaller or larger SV40 fragments mayalso be used, provided there is included the approximately 250-bpsequence extending from the HindIII site toward the BgII site located inthe viral origin of replication. Further, it is also possible, and oftendesirable, to utilize promoter or control sequences normally associatedwith the desired gene sequence, provided such control sequences arecompatible with the host cell systems.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may beprovided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter is oftensufficient.

Satisfactory amounts of protein are produced by cell cultures; however,refinements, using a secondary coding sequence, serve to enhanceproduction levels even further. One secondary coding sequence comprisesdihydrofolate reductase (DHFR) that is affected by an externallycontrolled parameter, such as methotrexate (MTX), thus permittingcontrol of expression by control of the methotrexate concentration.

In selecting a preferred host cell for transfection by the vectors ofthe invention that comprise DNA sequences encoding both VEGF and DHFRprotein, it is appropriate to select the host according to the type ofDHFR protein employed. If wild-type DHFR protein is employed, it ispreferable to select a host cell that is deficient in DHFR, thuspermitting the use of the DHFR coding sequence as a marker forsuccessful transfection in selective medium that lacks hypoxanthine,glycine, and thymidine. An appropriate host cell in this case is theChinese hamster ovary (CHO) cell line deficient in DHFR activity,prepared and propagated as described by Urlaub and Chasin, Proc. Natl.Acad. Sci. (USA) 77, 4216 (1980).

On the other hand, if DHFR protein with low binding affinity for MTX isused as the controlling sequence, it is not necessary to useDHFR-deficient cells. Because the mutant DHFR is resistant tomethotrexate, MTX-containing media can be used as a means of selectionprovided that the host cells are themselves methotrexate sensitive. Mosteukaryotic cells that are capable of absorbing MTX appear to bemethotrexate sensitive. One such useful cell line is a CHO line, CHO-K1(ATCC No. CCL 61).

b. Typical Methodology Employable

Construction of suitable vectors containing the desired coding andcontrol sequences employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in theform desired to prepare the plasmids required.

If blunt ends are required, the preparation may be treated for 15minutes at 15° C. with 10 units of Polymerase I (Klenow),phenol-chloroform extracted, and ethanol precipitated.

Size separation of the cleaved fragments may be performed using 6percent polyacrylamide gel described by Goeddel et al., Nucleic AcidsRes. 8, 4057 (1980).

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are typically used to transform E. coli K12 strain 294(ATCC 31,446) or other suitable E. coli strains, and successfultransformants selected by ampicillin or tetracycline resistance whereappropriate. Plasmids from the transformants are prepared and analyzedby restriction mapping and/or DNA sequencing by the method of Messing etal., Nucleic Acids Res. 9, 309 (1981) or by the method of Maxam et al.,Methods of Enzymology 65, 499 (1980).

After introduction of the DNA into the mammalian cell host and selectionin medium for stable transfectants, amplification of DHFR-protein-codingsequences is effected by growing host cell cultures in the presence ofapproximately 20,000-500,000 nM concentrations of methotrexate, acompetitive inhibitor of DHFR activity. The effective range ofconcentration is highly dependent, of course, upon the nature of theDHFR gene and the characteristics of the host. Clearly, generallydefined upper and lower limits cannot be ascertained. Suitableconcentrations of other folic acid analogs or other compounds thatinhibit DHFR could also be used. MTX itself is, however, convenient,readily available, and effective.

Other techniques employable are described in a section just prior to theexamples.

4. Utilities and Formulation

The variant VEGF antagonists of the present invention have a number oftherapeutic uses associated with the vascular endothelium. Such usesinclude, for example, incorporation into formed articles which can beused in modulating endothelial cell growth and angiogenesis. Inaddition, tumor invasion and metastasis may be modulated with thesearticles. Other disorders for which the polypeptides of the presentinvention may find use are discussed supra.

For the indications referred to above, the variant VEGF antagonistmolecule will be formulated and dosed in a fashion consistent with goodmedical practice taking into account the specific disease or disorder tobe treated, the condition of the individual patient, the site ofdelivery of the VEGF antagonist, the method of administration, and otherfactors known to practitioners. Thus, for purposes herein, the“therapeutically effective amount” of the VEGF is an amount that iseffective either to prevent, lessen the worsening of, alleviate, or curethe treated condition, in particular that amount which is sufficient tosubstantially inhibit the growth of vascular endothelium in vivo.

VEGF amino acid sequence variants and derivatives that areimmunologically crossreactive with antibodies raised against native VEGFare useful in immunoassays for VEGF as standards, or, when labeled, ascompetitive reagents.

The VEGF antagonist is prepared for storage or administration by mixingVEGF antagonist having the desired degree of purity with physiologicallyacceptable carriers, excipients, or stabilizers. Such materials arenon-toxic to recipients at the dosages and concentrations employed. Ifthe VEGF antagonist is water soluble, it may be formulated in a buffersuch as phosphate or other organic acid salt preferably at a pH of about7 to 8. If a VEGF variant is only partially soluble in water, it may beprepared as a microemulsion by formulating it with a nonionic surfactantsuch as Tween, Pluronics, or PEG, e.g., Tween 80, in an amount of0.04-0.05% (w/v), to increase its solubility.

Optionally other ingredients may be added such as antioxidants, e.g.,ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; and sugar alcohols such asmannitol or sorbitol.

The VEGF antagonist to be used for therapeutic administration must besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). The VEGF ordinarilywill be stored in lyophilized form or as an aqueous solution if it ishighly stable to thermal and oxidative denaturation. The pH of the VEGFantagonist preparations typically will be about from 6 to 8, althoughhigher or lower pH values may also be appropriate in certain instances.It will be understood that use of certain of the foregoing excipients,carriers, or stabilizers will result in the formation of salts of theVEGF antagonist.

If the VEGF antagonist is to be used parenterally, therapeuticcompositions containing the VEGF antagonist generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

Generally, where the disorder permits, one should formulate and dose theVEGF for site-specific delivery. This is convenient in the case ofsite-specific solid tumors.

Sustained release formulations may also be prepared, and include theformation of microcapsular particles and implantable articles. Forpreparing sustained-release VEGF antagonist compositions, the VEGFantagonist is preferably incorporated into a biodegradable matrix ormicrocapsule. A suitable material for this purpose is a polylactide,although other polymers of poly-(α-hydroxycarboxylic acids), such aspoly-D-(−)-3-hydroxybutyric acid (EP 133,988A), can be used. Otherbiodegradable polymers include poly(lactones), poly(acetals),poly(orthoesters), or poly(orthocarbonates). The initial considerationhere must be that the carrier itself, or its degradation products, isnontoxic in the target tissue and will not further aggravate thecondition. This can be determined by routine screening in animal modelsof the target disorder or, if such models are unavailable, in normalanimals. Numerous scientific publications document such animal models.

For examples of sustained release compositions, see U.S. Pat. No.3,773,919, EP 58,481 A, U.S. Pat. No. 3,887,699, EP 158,277A, CanadianPatent No. 1176565, U. Sidman et al., Biopolymers 22, 547 [1983], and R.Langer et al., Chem. Tech. 12, 98 [1982].

When applied topically, the VEGF antagonist is suitably combined withother ingredients, such as carriers and/or adjuvants. There are nolimitations on the nature of such other ingredients, except that theymust be pharmaceutically acceptable and efficacious for their intendedadministration, and cannot degrade the activity of the activeingredients of the composition. Examples of suitable vehicles includeointments, creams, gels, or suspensions, with or without purifiedcollagen. The compositions also may be impregnated into transdermalpatches, plasters, and bandages, preferably in liquid or semi-liquidform.

For obtaining a gel formulation, the VEGF antagonist formulated in aliquid composition may be mixed with an effective amount of awater-soluble polysaccharide or synthetic polymer such as polyethyleneglycol to form a gel of the proper viscosity to be applied topically.The polysaccharide that may be used includes, for example, cellulosederivatives such as etherified cellulose derivatives, including alkylcelluloses, hydroxyalkyl celluloses, and alkylhydroxyalkyl celluloses,for example, methylcellulose, hydroxyethyl cellulose, carboxymethylcellulose, hydroxypropyl methylcellulose, and hydroxypropyl cellulose;starch and fractionated starch; agar; alginic acid and alginates; gumarabic; pullullan; agarose; carrageenan; dextrans; dextrins; fructans;inulin; mannans; xylans; arabinans; chitosans; glycogens; glucans; andsynthetic biopolymers; as well as gums such as xanthan gum; guar gum;locust bean gum; gum arabic; tragacanth gum; and karaya gum; andderivatives and mixtures thereof. The preferred gelling agent herein isone that is inert to biological systems, nontoxic, simple to prepare,and not too runny or viscous, and will not destabilize the VEGFantagonist held within it.

Preferably the polysaccharide is an etherified cellulose derivative,more preferably one that is well defined, purified, and listed in USP,e.g., methylcellulose and the hydroxyalkyl cellulose derivatives, suchas hydroxypropyl cellulose, hydroxyethyl cellulose, and hydroxypropylmethylcellulose. Most preferred herein is methylcellulose.

The polyethylene glycol useful for gelling is typically a mixture of lowand high molecular weight polyethylene glycols to obtain the properviscosity. For example, a mixture of a polyethylene glycol of molecularweight 400-600 with one of molecular weight 1500 would be effective forthis purpose when mixed in the proper ratio to obtain a paste.

The term “water soluble” as applied to the polysaccharides andpolyethylene glycols is meant to include colloidal solutions anddispersions. In general, the solubility of the cellulose derivatives isdetermined by the degree of substitution of ether groups, and thestabilizing derivatives useful herein should have a sufficient quantityof such ether groups per anhydroglucose unit in the cellulose chain torender the derivatives water soluble. A degree of ether substitution ofat least 0.35 ether groups per anhydroglucose unit is generallysufficient. Additionally, the cellulose derivatives may be in the formof alkali metal salts, for example, the Li, Na, K, or Cs salts.

If methylcellulose is employed in the gel, preferably it comprises about2-5%, more preferably about 3%, of the gel and the VEGF antagonist ispresent in an amount of about 300-1000 mg per ml of gel.

The dosage to be employed is dependent upon the factors described above.As a general proposition, the VEGF antagonist is formulated anddelivered to the target site or tissue at a dosage capable ofestablishing in the tissue a VEGF antagonist level greater than about0.1 ng/cc up to a maximum dose that is efficacious but not unduly toxic.This intra-tissue concentration should be maintained if possible bycontinuous infusion, sustained release, topical application, orinjection at empirically determined frequencies.

5. Pharmaceutical Compositions

The compounds of the present invention can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebythe VEGF antagonists hereof are combined in admixture with apharmaceutically acceptable carrier vehicle. Suitable carrier vehiclesand their formulation, inclusive of other human proteins, e.g., humanserum albumin, are described, for example, in Remington's PharmaceuticalSciences, 16th ed., 1980, Mack Publishing Co., edited by Oslo et al. thedisclosure of which is hereby incorporated by reference. The VEGFvariants herein may be administered parenterally, or by other methodsthat ensure its delivery to the bloodstream in an effective form.

Compositions particularly well suited for the clinical administration ofthe VEGF antagonists hereof employed in the practice of the presentinvention include, for example, sterile aqueous solutions, or sterilehydratable powders such as lyophilized protein. It is generallydesirable to include further in the formulation an appropriate amount ofa pharmaceutically acceptable salt, generally in an amount sufficient torender the formulation isotonic. A pH regulator such as arginine base,and phosphoric acid, are also typically included in sufficientquantities to maintain an appropriate pH, generally from 5.5 to 7.5.Moreover, for improvement of shelf-life or stability of aqueousformulations, it may also be desirable to include further agents such asglycerol. In this manner, variant t-PA formulations are renderedappropriate for parenteral administration, and, in particular,intravenous administration.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. For example, “bolus” doses may typically be employed withsubsequent administrations being given to maintain an approximatelyconstant blood level, preferably on the order of about 3 μg/ml.

However, for use in connection with emergency medical care facilitieswhere infusion capability is generally not available and due to thegenerally critical nature of the underlying disease, it will generallybe desirable to provide somewhat larger initial doses, such as anintravenous bolus.

For the various therapeutic indications referred to for the compoundshereof, the VEGF antagonists will be formulated and dosed in a fashionconsistent with good medical practice taking into account the specificdisorder to be treated, the condition of the individual patient, thesite of delivery, the method of administration and other factors knownto practitioners in the respective art. Thus, for purposes herein, the“therapeutically effective amount” of the VEGF molecules hereof is anamount that is effective either to prevent, lessen the worsening of,alleviate, or cure the treated condition, in particular that amountwhich is sufficient to substantially reduce or inhibit the growth ofvascular endothelium in vivo. In general a dosage is employed capable ofestablishing in the tissue that is the target for the therapeuticindication being treated a level of a VEGF antagonist hereof greaterthan about 0.1 ng/cm³ up to a maximum dose that is efficacious but notunduly toxic. It is contemplated that intra-tissue administration may bethe choice for certain of the therapeutic indications for the compoundshereof.

The following examples are intended merely to illustrate the best modenow known for practicing the invention but the invention is not to beconsidered as limited to the details of such examples.

EXAMPLE I

Materials—Muta-gene phagemid in vitro mutagenesis kit, horse-radishperoxidase conjugated goat IgG specific for murine IgG, pre-stainedlow-range MW standards and Trans-Blot Transfer Medium (purenitrocellulose membrane) were purchased from BioRad Laboratories(Richmond, Calif.). Qiagen plasmid Tip 100 kit and Sequenase version 2.0were from Qiagen (Chatsworth, Calif.) and United States Biochemical(Cleveland, Ohio), respectively. SDS gels (4-20% gradientpolyacrylamide) and pre-cut blotting paper were from IntegratedSeparations Systems (Natick, Mass.). SDS sample buffer (x concentrate)and various restriction enzymes were from New England Biolabs (Beverly,Mass.). O-phenylenediamine, citrate phosphate buffers, sodium dodecylsulfate, and H₂O₂ substrate tablets were purchased from Sigma (St.Louis, Mo.). BufferEZE formula 1 (transfer buffer) and X-OMat AR X-rayfilm were from Eastman Kodak Co. (Rochester, N.Y.). Maxosorb andImmunlon-1 microtiter plates were purchased from Nunc (Kamstrup,Denmark) and Dynatech (Chantilly, Va.), respectively. Cell cultureplates (12-well) and culture media (with calf serum) were from Costar(Cambridge, Mass.) and Gibco (Grand Island, N.Y.), respectively.Polyethylene-20-sorbitan monolaurate (Tween-20) was from Fisher Biotech(Fair Lawn, N.J.). G25 Sephadex columns (PD-10) and ¹²⁵I labeled ProteinA were from Pharmacia (Piscataway, N.J.) and Amersham (ArlingtonHeights, Ill.), respectively. Bovine serum albumin (BSA) and rabbit IgGanti-human IgG (Fc-specific) were purchased from Cappel (Durham, N.C.)and Calbiochem (La Jolla, Calif.), respectively. Plasmid vector (pRK5),competent E. coli cells (DH5a and CJ236), synthetic oligonucleotides,cell culture medium, purified CHO-derived VEGF₁₆₅, monoclonal (MatesA4.6.1, 2E3, 4D7, SC3, and SF8) and polyclonal antibodies to VEGF₁₆₅were prepared at Genentech, Inc. (South San Francisco, Calif.).Construction, expression and purification of FLT-1, flkI and KDRreceptor-IgG chimeras was as described by Park, et al. J. Biol. Chem.269, 25646-25654 (1994).

Site-directed Mutagenesis and Expression of VEGF Variants—Site-directedmutagenesis was performed using the Muta-Gene Phagemid in vitromutagenesis kit according to the method of Kunkel Proc. Natl. Acad. Sci.82, 488-492 (1985) and Kunkel et al., Methods Enzymol. 154, 367-382(1987). A plasmid vector pRK5 containing cDNA for VEGF₁₆₅ isoform wasused for mutagenesis and transient expression. The pRK5 vector is amodified pUC118 vector and contains a CMV enhancer and promoter[Nakamaye et al., Nucleic Acids Res. 14, 9679-9698 (1986) and Vieira etal., Methods Enzymol. 155, 3-11 (1987)]. The mutagenized DNA waspurified using the Qiagen Plasmid Midi Kit Tip 100 and the sequence ofthe mutations was verified using Sequenase Version 2.0 Kit. The mutatedDNA was analyzed by restriction enzyme digestion as described bySambrook, et al., Molecular Cloning: A Laboratory Manual part I,C5.28-5.32, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989).

Transient transfection of human fetal kidney “293 cells” was performedin 6-well plates using the modified calcium phosphate precipitate methodas previously described [Jordan et al., Bio/Technology (manuscript inpreparation) (1994); Chen et al., Mol. Cell. Biol. 7, 2745-2752 (1987);Gorman et al., DNA and Protein Engineering Techniques 2, 3-10 (1990);Graham et al., Virology 52, 456-467 (1973)]. Briefly, approximately1.2×10⁶ cells were incubated overnight at 37° C. in the presence of 15μg of precipitated DNA. Cell culture supernatant was replaced with serumfree medium, and cell monolayers were incubated for 72 hours at 37° C.Conditioned media (3 ml) was harvested, centrifuged, aliquoted andstored at −70° C. until use.

Quantitation of VEGF₁₆₅ Variants by ELISA—A radioimmunometric assaypreviously described [Aiello et al., N. Engl. J. Med. 331, 1480-1487(1994)], was adapted for the quantitation of VEGF mutants by thefollowing procedure. Individual wells of a 96-well microtiter plate werecoated with 100 μl of a 3 μg/ml solution of an anti-VEGF₁₆₅ polyclonalantibody in 50 mM sodium carbonate buffer pH 9.6 overnight at 4° C. Thesupernatant was discarded, and the wells were washed 4 times with PBScontaining 0.03% Tween 80. The plate was blocked in assay buffer (0.5%BSA, 0.03% Tween 80, 0.01% Thimerosal in PBS) for one hr (300 μl/well)at ambient temperature, then the wells were washed. Diluted samples (100μl) and VEGF₁₆₅ standard (ranging from 0.1 to 10 ng/ml) were added toeach well and incubated for one hr at ambient temperature with gentleagitation. The supernatant was discarded, and the wells were washed.Anti-VEGF murine monoclonal antibody 5F8 solution (100 μl at 1 μg/ml)was added, and the microtiter plate was incubated at ambient temperaturefor one hr with gentle agitation. After the supernatant was discarded,the plate washed and horseradish peroxidase conjugated goat IgG specificfor murine IgG (100 μl) at a dilution of 1:25000 was immediately addedto each well. The plate was incubated for one hr at ambient temperaturewith gentle agitation after which the supernatant discarded, the wellswashed, and developed by addition of ortho-phenylenediamine (0-04%),H₂O₂ (0.012%) in 50 mM citrate phosphate buffer pH 5 (100 μl), thenincubated in the dark at ambient temperature for 10 min. The reactionwas stopped by adding 50 μl of 4.5 N H₂SO₄ to each well and theabsorbance was measured at 492 nm on a microplate reader (SLT Labs). Theconcentrations of VEGF₁₆₅ variants were quantitated by interpolation ofa standard curve using non-linear regression analysis. For purposes ofcomparison, a second ELISA was developed that utilized a dual monoclonalformat. The assay was similar to the above described ELISA, except aneutralizing monoclonal antibody (Mab A4.6.1) was used to coat themicrotiter plates [Kim et al., Growth Factors 7, 53-64 (1992)].

Immunoblotting of VEGF mutants—Aliquots of conditioned cell media (16μl) containing VEGF or VEGF mutant (approx. 10 ng) were added to x SDSsample buffer (4 μl) and heated at 90° C. for 3 min prior to loading onSDS polyacrylamide (4 to 20% acrylamide) gels. Pre-stained MW standards(10 μl) were loaded in the outer lanes of the SDS gels. Gels were run at25 mA for 90 min at 4° C. Gels were transferred to nitrocellulose paperin a Bio-Rad tank blotter containing BufferEZE with 0.1% SDS for 90 minat 250 mA at 25° C. Nitrocellulose was pre-wetted in transfer bufferwith 0.1% SDS for 10 min prior to use. Transferred immunoblots wereblocked in PBS overnight with 1.0% BSA and 0.1% Tween 20 (blockingbuffer) at 4° C. A solution containing 5 murine anti-VEGF Mabs (A.4.6.1,5C3, 5F8, 4D7, and 2E3) was prepared with 2 μg/ml of each Mab inblocking buffer and used as primary antibody. The primary antibodysolution was incubated with the immunoblots for 4 hr at 25° C. withgentle agitation, then washed 3× for 10 min in blocking buffer at 25° C.¹²⁵I labeled Protein A was diluted to 10⁴ cpm/ml (final concentration)in blocking buffer and incubated with the immunoblots for 60 min withgentle agitation at 25° C. Immunoblots were washed 3× for 10 min inblocking buffer at 25° C., then dried on filter paper and placed onKodak X-Omat film with two intensifying screens at −70° C. for 3 days.

Preparation of ¹²⁵I labeled VEGF₁₆₅—Radiolabeling of CHO-derived VEGF₁₆₅was prepared using a modification of the chloramine T catalyzediodination method [Hunter et al., Nature 194, 495-496 (1962)]. In atypical reaction, 10 μl of 1 M Tris-HCl, 0.01% Tween 20 at pH 7.5 wasadded to 5 μl of sodium iodide-125 (0.5 milliCuries, 0.24 nmol) in acapped reaction vessel. To this reaction, 10 μl of CHO-derived VEGF₁₆₅(10 μg, 0.26 nmol) was added. The iodination was initiated by additionof 10 μl of 1 mg/ml chloramine T in 0.1 M sodium phosphate, pH 7.4.After 60 sec, iodination was terminated by addition of sodiummetabisulfite (20 μl, 1 mg/ml) in 0.1 M sodium phosphate, pH 7.5. Thereaction vessel was vortexed after each addition. The reaction mixturewas applied to a PD-10 column (G25 Sephadex) that was pre-equilibratedwith 0.5% BSA, 0.01% Tween 20 in PBS. Fractions were collected andcounted for radioactivity with a gamma scintillation counter (LKB model1277). Typically, the specific radioactivity of the iodinated VEGF was26.+−.2.5 μCi/μg, which corresponded to one ¹²⁵I per two molecules ofVEGF₁₆₅ dimer.

VEGF₁₆₅ Receptor Binding Assay—The assay was performed in 96-wellimmunoplates (Immulon-1); each well was coated with 100 μl of a solutioncontaining 10 μg/ml of rabbit IgG anti-human IgG (Fc-specific) in 50 mMsodium carbonate buffer pH 9.6 overnight at 4° C. After the supernatantwas discarded, the wells were washed three times in washing buffer(0.01% Tween 80 in PBS). The plate was blocked (300 μl/well) for one hrin assay buffer (0.5% BSA, 0.03% Tween 80, 0.01% Thimerosal in PBS). Thesupernatant was discarded and the wells were washed. A cocktail wasprepared with conditioned cell media containing VEGF₁₆₅ mutants atvarying concentrations (100 μl), ¹²⁵I radiolabeled VEGF₁₆₅ (approx.5×10₃ cpm in 50 μl) which was mixed with VEGF receptor-IgG chimericprotein, FLT-1 IgG, flk-1 IgG or KDR-IgG (3-15 ng/ml, finalconcentration, 50 μl) in micronic tubes. Aliquots of this solution (100μl) were added to pre-coated microtiter plates and incubated for 4 hr atambient temperature with gentle agitation. The supernatant wasdiscarded, the plate washed, and individual microtiter wells werecounted by gamma scintigraphy (LKB model 1277). The competitive bindingbetween unlabeled VEGF₁₆₅ (or VEGF₁₆₅ mutants) and ¹²⁵I radiolabeledVEGF₁₆₅ to the FLT-1, Flk-1, or KDR receptors were plotted, and analyzedusing a four parameter fitting program (Kaleidagraph, AdelbeckSoftware).

The apparent dissociation constant for each VEGF mutant was estimatedfrom the concentration required to achieve 50% inhibition (IC₅₀).

Assay for Vascular Endothelial Cell Growth—The mitogenic activity ofVEGF variants was determined by using bovine adrenal corticalendothelial (ACE) cells as target cells as previously described [Ferraraet al., Biochem. Biophys. Res. Comm. 161, 851-859 (1989)]. Briefly,cells were plated sparsely (7000 cells/well) in 12 well plates andincubated overnight in Dulbecco's modified Eagle's medium supplementedwith 10% calf serum, 2 mM glutamine, and antibiotics. The medium wasexchanged the next day, and VEGF or VEGF mutants, diluted in culturemedia at concentrations ranging from 100 ng/ml to 10 pg/ml, were layeredin duplicate onto the seeded cells. After incubation for 5 days at 37°C., the cells were dissociated with trypsin, and quantified using aCoulter counter.

Isolation of VEGF cDNA

Total RNA was extracted [Ullrich et al., Science 196, 1313-1317 (1977)]from bovine pituitary follicular cells [obtained as described by Ferraraet al., Meth. Enzymol. supra, and Ferrara et al., Am. J. Physiol.,supra] and the polyadenylated mRNA fraction was isolated byoligo(dT)-cellulose chromatography. Aviv et al., Proc. Natl. Acad. Sci.USA 69, 1408-1412 (1972). The cDNA was prepared [Wickens et al., J.Biol. Chem. 253, 2483-2495 (1978)] by priming with dT₁₂₋₁₈ or a randomhexamer dN₆.

The double-stranded cDNA was synthesized using a cDNA kit from Amersham,and the resulting cDNA was subcloned into EcoRI-cleaved Igt10 asdescribed [Huynh et al., DNA Cloning Techniques, A Practical Approach,Glover ed. (IRL, Oxford, 1985)], except that asymmetric EcoRI linkers[Norris et al., Gene 7, 355-362 (1979)] were used, thus avoiding theneed for the EcoRI methylase treatment.

The recombinant phage were plated on E. coli C600 Hfl [Huynh et al.supra] and replica plated onto nitrocellulose filters. Benton et al.,Science 196, 180-182 (1977). These replica were hybridized with a³²P-labeled [Taylor et al., Biochim. Biophys. Acta, 442, 324-330 (1976)]synthetic oligonucleotide probe of the sequence:5′-CTATGGCTGAAGGCGGCCAGAAGCCTCACGAAGTGGTGAAGTTCATGGACGT GTATCA-3′ (SEQ.ID NO:1) at 42° C. in 20% formamide, 5×SSC, 50 mM sodium phosphate pH6.8, 0.1% sodium pyrophosphate, 5× Denhardt's solution, and 50 mg/mlsalmon sperm DNA, and washed in 2×SSC, 0.1% SDS at 42° C.

One positive clone, designated I.vegf.6, was identified. This clone,labeled with ³²P, was used as a probe to screen an oligo-dT-primed humanplacenta cDNA library, and positive clones were observed. When a humanpituitary cDNA library was screened with the same labeled clone, nopositive clones were detected.

The complete nucleotide sequence of the clone I.vegf.6 was determined bythe dideoxyoligonucleotide chain termination method [Sanger et al.,Proc. Natl. Acad. Sci. USA 74, 5463-5467 (1977)] after subcloning intothe pRK5 vector. The sequence obtained, along with the imputed aminoacid sequence, including the signal sequence.

Expression of VEGF-Encoding Gene in Mammalian Cells

The final expression vector, pRK5.vegf.6, was constructed from I.vegf.6and pRK5. The construction of pRK5 and pRK5.vegf.6 is described below indetail.

A. Construction of pRK5

A.1. Construction of pF8CIS

The initial three-part construction of the starting plasmid pF8CIS isdescribed below.

1) The ampicillin resistance marker and replication origin of the finalvector was derived from the starting plasmid pUC13 pML, a variant of theplasmid pML (Lusky, M. and Botchen, M., Nature, 293, 79 [1981]). pUC13pML was constructed by transferring the polylinker of pUC13 (Vieira, J.and Messing, J., Gene, 19, 259 (1982)) to the EcoRI and HindIII sites ofpML. A second starting plasmid pUC8-CMV was the source of the CMVenhancer, promoter and splice donor sequence. pUC8-CMV was constructedby inserting approximately 800 nucleotides for the CMV enhancer,promoter and splice donor sequence into the blunted PstI and SphI sitesof pUC8. Vieira, J. and Messing, J., op. cit. Synthetic BamHI-HindIIIlinkers (commercially available from New England Biolabs) were ligatedto the cohesive BamHI end creating a HindIII site. Following thisligation a HindIII-HincII digest was performed. This digest yielded afragment of approximately 800 bp that contained the CMV enhancer,promoter and splice donor site. Following gel isolation, this 800 bpfragment was ligated to a 2900 bp piece of pUC13pML. The fragmentrequired for the construction of pF8CIS was obtained by digestion of theabove intermediate plasmid with SalI and HindIII. This 3123 bp piececontained the resistance marker for ampicillin, the origin ofreplication from pUC13pML, and the control sequences for the CMV,including the enhancer, promoter, and splice donor site.

2) The Ig variable region intron and splice acceptor sequence wasconstructed using a synthetic oligomer. A 99 mer and a 30 mer werechemically synthesized having the following sequence for the IgG intronand splice acceptor site (Bothwell et al., Nature, 290, 65-67 [1981]):(SEQ. ID NO: 2)  1 5′ AGTAGCAAGCTTGACGTGTGGCAGGCTTGA . . . (SEQ. ID NO:3) 31 GATCTGGCCATACACTTGAGTGACAATGA . . . (SEQ. ID NO: 4) 60CATCCACTTTGCCTTTCTCTCCACAGGT . . . (SEQ. ID NO: 5) 88 GTCCACTCCCAG 3′(SEQ. ID NO: 6)  1 3′ CAGGTGAGGGTGCAGCTTGACGTCGTCGGA 5′DNA polymerase I (Klenow fragment) filled in the synthetic piece andcreated a double-stranded fragment. Wartell, R. M. and W. S. Reznikoff,Gene, 9, 307 (1980). This was followed by a double digest of PstI andHindIII. This synthetic linker was cloned into pUC13 (Veira and Messing,op. cit.) at the PstI and HindIII sites. The clones containing thesynthetic oligonucleotide, labeled pUCIg.10, was digested with PstI. AClaI site was added to this fragment by use of a PstI-ClaI linker.Following digestion with HindIII a 118-bp piece containing part of theIg intron and the Ig variable region splice acceptor was gel isolated.

3) The third part of the construction scheme replaced the hepatitissurface antigen 3′ end with the polyadenylation site and transcriptiontermination site of the early region of SV40. A vector, pUC.SV40,containing the SV40 sequences was inserted into pUC8 at the BamHI sitedescribed by Vieira and Messing, op. cit. pUC.SV40 was then digestedwith EcoRI and HpaI. A 143 bp fragment containing the SV40polyadenylation sequence was gel isolated from this digest. Twoadditional fragments were gel isolated following digestion of pSVE.8c1D.(European Pat. Pub. No. 160,457). The 4.8 kb fragment generated by EcoRIand Cla1 digestion contains the SV40-DHFR transcription unit, the originof replication of pML and the ampicillin resistance marker. The 7.5-kbfragment produced following digestion with ClaI and HpaI contains thecDNA for Factor VIII. A three-part ligation yielded pSVE.8c24D. Thisintermediate plasmid was digested by ClaI and SalI to give a 9611 bpfragment containing the cDNA for Factor VIII with an SV40 poly A sitefollowed by the SV40 DHFR transcription unit.

The final three-part ligation to yield pF8CIS used: a) the 3123 bpSalI-HindIII fragment containing the origin of replication, theampicillin resistance marker, and the CMV enhancer, promoter, and splicedonor site; b) the 118 bp HindIII-ClaI fragment containing the Ig intronand splice acceptor site; and c) a 9611 bp ClaI-SalI fragment containingthe cDNA for Factor VIII, the SV40 polyadenylation site, and the SV40DHFR transcription unit.

A.2. Construction of pCIS2.8c28D

pCIS2.8c28D comprises a 90 kd subunit of Factor VIII joined to a 73 kdsubunit of Factor VIII. The 90 kd comprises amino acids 1 through 740and the 73 kd subunit amino acids 1690 through 2332. This construct wasprepared by a three-part ligation of the following fragments: a) the12617-bp ClaI-SstII fragment of pF8CIS (isolated from a dam-strain andBAP treated); b) the 216-bp SstII-PstI fragment of pF8CIS; and c) ashort PstI-ClaI synthetic oligonucleotide that was kinased.

Two different fragments, A and B, were cloned into the same pUC118BamHI-PstI BAP vector. The A fragment was the 408 bp BamHI-HindIIIfragment of pUC408BH and the B fragment was a HindIII-PstIoligonucleotide. This oligonucleotide was used without kinasing toprevent its polymerization during ligation.

After ligation of the A and B fragments into the vector, the expectedjunction sequences were confirmed by DNA sequencing of the regionsencompassed by the nucleotides.

The resulting plasmid, pCIS2.8c28D, was constructed with a four-partligation. The fusion plasmid was cut with BamHI and PstI and the 443 bpfragment isolated. The remaining three fragments of the four-partligation were: 1) 1944 bp ClaI-BamHI of pSVEFVIII (European Pat. Publ.No. 160,457); 2) a 2202 bp BamHI-XbaI fragment of pSVEFVIII, which wasfurther partially digested with PstI and the 1786 bp PstI-XbaI fragmentwas isolated, and 3) the 5828 bp XbaI-ClaI BAP fragment of pCIS2.8c24D.The translated DNA sequence of the resultant variant in the exact fusionjunction region of pCIS2.8c28D was determined and correlates.

A.3. Construction of pRK5

The starting plasmid for construction of pRK5 was pCIS2.8c28D. The basenumbers in paragraphs 1 through 6 refer to pCIS2.8c28D with base one ofthe first T of the EcoRI site preceding the CMV promoter. Thecytomegalovirus early promoter and intron and the SV40 origin and polyAsignal were placed on separate plasmids.

1. The cytomegalovirus early promoter was cloned as an EcoRI fragmentfrom pCIS2.8c28D (9999-1201) into the EcoRI site of pUC118 describedabove. Twelve colonies were picked and screened for the orientation inwhich single-stranded DNA made from pUC118 would allow for thesequencing from the EcoRI site at 1201 to the EcoRI site at 9999. Thisclone was named pCMVE/P.

2. Single-stranded DNA was made from pCMVE/P in order to insert an SP6(Green, M R et al., Cell 32, 681-694 [1983]) promoter by site-directedmutagenesis. A synthetic 110 mer that contained the sequences from −69to +5 of SP6 promoter (see Nucleic Acids Res., 12, 7041 [1984]) wereused along with 18-bp fragments on either end of the oligomercorresponding to the CMVE/P sequences. Mutagenesis was done by standardtechniques and screened using a labeled 110 mer at high and lowstringency. Six potential clones were selected and sequenced. A positiveclone was identified and labeled pCMVE/PSP6.

3. The SP6 promoter was checked and shown to be active, for example, byadding SP6 RNA polymerase and checking for RNA of the appropriate size.

4. A Cla-NotI-Sma adapter was synthesized to encompass the location fromthe ClaI site (912) to the SmaI site of pUC118 in pCMVE/P (step 1) andpCMVE/PSP6 (step 2). This adapter was ligated into the ClaI-SmaI site ofpUC118 and screened for the correct clones. The linker was sequenced inboth and clones were labeled pCMVE/PSP6-L and pCMVE/P-L.

5. pCMVE/PSP6-L was cut with SmaI (at linker/pUC118 junction) andHindIII (in pUC 118). A Hpal (5573)-to-HindIII (6136) fragment frompSVORAADRI11, described below, was inserted into SmaI-HindIII ofpCMVE/PSP6-L. This ligation was screened and a clone was isolated andnamed pCMVE/PSP6-L-SVORAADRI.

a) The SV40 origin and polyA signal was isolated as the XmnI(5475)-HindIII (6136) fragment from pCIS2.8c28D and cloned into theHindIII to SmaI sites of pUC119 (described in Vieira and Messing, op.cit.). This clone was named pSVORAA.

b) The EcoRI site at 5716 was removed by partial digestion with EcoRIand filling in with Klenow. The colonies obtained from self-ligationafter fill-in were screened and the correct clone was isolated and namedpSVORAADRI 11. The deleted EcoRI site was checked by sequencing andshown to be correct.

c) The HpaI (5573) to HindIII (6136) fragment of pSVORAADRI 11 wasisolated and inserted into pCMVE/PSP6-L (see 4 above).

6. pCMVE/PSP6-L-SVOrAADRI (step 5) was cut with EcoRI at 9999, bluntedand self-ligated. A clone without an EcoRI site was identified and namedpRK.

7. pRK was cut with SmaI and BamHI. This was filled in with Klenow andrelegated. The colonies were screened. A positive clone was identifiedand named pRKDBam/Sma3.

8. The HindIII site of pRKDBam/Sma3 was converted to a HpaI site using aconverter. (A converter is a piece of DNA used to change one restrictionsite to another. In this case one end would be complementary to aHindIII sticky end and the other end would have a recognition site forHpaI.) A positive clone was identified and named pRKDBam/Sma, HIII-HpaI1.

9. pRKDBam/Sma, Hil-HpaI 1 was cut with PstI and NotI and anEcoRI-HindIII linker and HindIII-EcoRI linker were ligated in. Clonesfor each linker were found. However, it was also determined that toomany of the HpaI converters had gone in (two or more converters generatea PvuII site). Therefore, these clones had to be cut with HpaI andself-ligated.

10. RI-HIII clone 3 and HIII-RI clone 5 were cut with HpaI, diluted, andself-ligated. Positives were identified. The RI-HIII clone was namedpRK5.

B. Construction of pRK5.vegf.6

The clone I.vegf.6 was treated with EcoRI and the EcoRI insert wasisolated and ligated into the vector fragment of pRK5 obtained bydigestion of pRK5 with EcoRI and isolation of the large fragment. Thetwo-part ligation of these fragments yielded the expression vector,pRK5.vegf.6, which was screened for the correct orientation of theVEGF-encoding sequence with respect to the promoter.

Further details concerning the construction of the basic pRK5 vector canbe taken from U.S. Pat. No. 5,332,671 that issued on 26 Jul. 1994, saidpatent being expressly incorporated herein by reference.

EXAMPLE 2

The following example details the methodology generally employed toprepare the various VEGF mutants covered by the present invention. Thebasic expression vector was prepared as follows:

Vector SDVF₁₆₅ containing the cDNA of VEGF₁₆₅ was obtained. The cDNA forVEGF₁₆₅ was isolated from SDVF₁₆₅ by restriction digestion with Hind IIIand Eco RI. This isolated insert was ligated into the pRK5 plasmidtaking advantage to the existence therein of Eco RI and Hind III sites.The resultant plasmid was transformed into competent CJ236 E. coli cellsto make a template for site-directed mutagenesis. The correspondingoligonucleotide containing the mutated site was then prepared—seeinfra—and the in vitro site-directed mutagenesis step was conducted inaccordance with known procedures using the BioRad Muta-Gene mutagenesiskit. After sequencing to determine that the mutagenized site wasincorporated into the final expression vector, the resultant vector wastransfected into 293 human kidney cells for transient expression.

The following oligonucleotides were prepared in order to make the finalmutated product. TABLE 1 Mutation 5′ to 3′ Sequence C51DCAGGGGCACATCGGATGGCTTGAA (SEQ ID NO:7) C51A CAGGGGCACGGCGGATGGCTTGAA(SEQ ID NO:8) C60D GTCATTGCAATCGCCCCCGCATCG (SEQ ID NO:9) C60AGTCATTGCAGGCGCCCCCGCATCG (SEQ ID NO:10) C51A, C60AGTCATTGCAGGCGCCCCCGCATCGCATCAGGGGCACGGC GGATGGCTTGAA (SEQ ID NO:11)C51D, C60D GTCATTGCAATCGCCCCCGCATCGCATCAGGGGCACATC GGATGGCTTGAA (SEQ IDNO:12)

Thus prepared in accordance with the insertion of the oligonucleotidesset forth in Table 1 above, left column there are prepared at thecorresponding mutation in the VEGF molecule in accordance with thenotation given under the left hand column entitled “Mutation”. Thenaming of the compound is in accord with naming convention. Thus, forthe first entry the mutation is referred to as “C51D”. This means thatat the 51 amino acid position of the VEGF molecule the cysteine (C)residue was mutated so as to insert an aspartic acid (D) at that 51position.

FIG. 2 is a diagram showing the native VEGF dimer and certain of thevariant VEGF polypeptides of the present invention. As shown in FIG. 2,the native VEGF molecule dimerizes through the formation of disulfidebonds between the cysteine at amino acid position 51 on one monomer andthe cysteine at amino acid position 60 on the other monomer and viceversa. Changing the cysteine residue at amino acid position 51 or 60 toaspartic acid (C51 D or C60D, respectively) prevents proper dimerizationand the formation of staggered dimer molecules. Changing both cysteineresidues at amino acid positions 51 and 60 (C51D, C60D) prevents dimerformation altogether.

Binding of VEGF Variants to VEGF Receptors—Native VEGF dimer and theVEGF variant polypeptides shown in FIG. 2 were tested for the ability tobind to the KDR and FLT-1 receptors. Receptor binding assays wereperformed as described above. The results obtained for binding to theKDR receptor are presented in FIGS. 3 and 4.

As shown in FIG. 3, all of the three VEGF variant polypeptides testedretained the ability to bind to the KDR receptor, although noneexhibited a binding affinity as great as the native VEGF dimer protein.The results presented in FIG. 3 also demonstrate that the monomericvariant polypeptide C51D, C60D retains the ability to bind to the KDRreceptor, however, it does so with a reduced binding affinity ascompared to the native dimer or two staggered dimers tested. FIG. 4demonstrates that the binding affinity of the C51D, C60D monomericvariant for the KDR receptor is approximately 500-fold less than thenative dimeric VEGF protein. Thus, these results demonstrate that eachof the VEGF variant polypeptides tested retain the ability to bind tothe KDR receptor, although at a lower binding affinity.

FIGS. 5 and 6 show the results obtained when measuring the binding ofthe polypeptides of FIG. 2 to the FLT-1 receptor. The results presentedin FIG. 5 demonstrate that all of the variants tested retain the abilityto bind to the FLT-1 receptor, although at reduced binding affinities ascompared to the native VEGF dimer. FIG. 6 demonstrates that the bindingaffinity of the C51D, C60D monomeric variant is approximately 140-lessfor the FLT-1 receptor than exhibited by the native VEGF dimer. Thus,these results demonstrate that each of the VEGF variant polypeptidestested retain the ability to bind to the FLT-1 receptor, although at alower binding affinity.

Stimulation of Mitogenesis by VEGF and Variants Thereof—Because the VEGFvariants shown in FIG. 2 were shown above to be capable of binding toboth the KDR and FLT-1 receptors, these variants were also tested fortheir ability to stimulate mitogenesis in endothelial cells. Themitogenic stimulation assays were performed as described above. Theresults from these assays are presented in FIG. 7.

As is shown in FIG. 7, while the native VEGF dimer molecule is capableof efficiently stimulating mitogenesis in endothelial cells, the VEGFvariants tested (staggered dimers C51D and C60D as well as the monomericvariant C51D, C60D) exhibit an inhibitory effect on the mitogenicstimulation of endothelial cells. These results demonstrate that properdimerization between the cysteine residues at amino acid positions 51and 60 of the native VEGF polypeptide is essential for efficientmitogenic stimulation of endothelial cells. As such, these datademonstrate that amino acid modifications which disrupt the ability ofVEGF monomeric units to properly dimerize function to inhibit themitogenic activity of the molecule. Given that these variant moleculeare capable of binding to and occupying the VEGF receptors withoutinducing a “native-VEGF-like” mitogenic response, such variant moleculesmay serve as effective antagonists of VEGF activity.

Ability of the C51D, C60D Monomer to Inhibit VEGF-Induced EndothelialCell Growth—The C51 D, C60D monomer polypeptide was employed in assaysdesigned to measure the ability of the monomer to inhibit theVEGF-induced growth of endothelial cells. Briefly, endothelial cellswere cultured in the presence of 3 ng/ml VEGF and varying amounts ofeither the A461 anti-VEGF monoclonal antibody or the C51D, C60D monomerpolypeptide. The results demonstrating the inhibitory effects of eachinhibitor on endothelial cell growth are presented in FIG. 8.

The results presented in FIG. 8 demonstrate that both the A461 anti-VEGFmonoclonal antibody and the C51D, C60D monomer polypeptide exhibitsubstantial inhibitory effects on VEGF-induced endothelial cell growth.These inhibitory effects increase as the ratio of inhibitor to VEGFincreases. As such, the C51D, C60D monomer polypeptide functions toinhibit the endothelial growth activating effect of VEGF.

CONCLUDING REMARKS

The foregoing description details specific methods which can be employedto practice the present invention. Having detailed such specificmethods, those skilled in the art will well enough know how to devisealternative reliable methods at arriving at the same information inusing the fruits of the present invention. Thus, however detailed theforegoing may appear in text, it should not be construed as limiting theoverall scope thereof; rather, the ambit of the present invention is tobe determined only by the lawful construction of the appended claims.All documents cited herein are hereby expressly incorporated byreference.

1. A VEGF antagonist molecule comprising a variant vascular endothelialgrowth factor polypeptide, said variant polypeptide comprising an aminoacid modification of at least one cysteine residue, wherein said aminoacid modification inhibits the ability of said variant polypeptide toproperly dimerize with another vascular endothelial growth factorpolypeptide monomer, wherein said antagonist molecule is capable ofbinding to vascular endothelial growth factor receptors withoutsignificantly inducing a vascular endothelial growth factor response,and functional derivatives of said antagonist molecule.
 2. Theantagonist molecule according to claim 1 wherein said amino acidmodification is a substitution of said at least one cysteine residuewith a different amino acid which is incapable of participating in adisulfide bond.
 3. The antagonist molecule according to claim 2 whereinsaid substitution is of the cysteine residue at amino acid position 51and/or 60 of the native VEGF amino acid sequence.
 4. The antagonistmolecule according to claim 3 wherein aspartic acid is substituted forcysteine.
 5. The antagonist molecule according to claim 4 comprising thesubstitution C51 D.
 6. The antagonist molecule according to claim 4comprising the substitution C60D.
 7. The antagonist molecule accordingto claim 1 wherein said amino acid modification is a chemicalmodification of said at least one cysteine residue which renders saidcysteine residue incapable of participating in a disulfide bond.
 8. Theantagonist molecule according to claim 7 wherein said chemicalmodification is of the cysteine residue at amino acid position 51 and/or60 of the native VEGF amino acid sequence.
 9. The antagonist moleculeaccording to claim 1 containing further amino acid modifications that donot otherwise affect the essential biological characteristics.
 10. Anisolated nucleic acid sequence comprising a sequence that encodes theVEGF antagonist molecule of claim
 1. 11. A replicable expression vectorcapable in a transformant host cell of expressing the nucleic acid ofclaim
 10. 12. Host cells transformed with the vector according to claim11.
 13. Host cells according to claim 12 which are Chinese hamster ovarycells.
 14. A composition of matter comprising the VEGF antagonistmolecule according to claim 1 compounded with a pharmaceuticallyacceptable carrier.
 15. A method of treatment which comprisesadministering a composition according to claim 14.